Stage-specific embryonic antigen (SSEA) glycosphingolipids (GSLs) found in the central nervous system are impficated in regulating cell-cell recognition, targeting and migration of cells during development. Through the action of fucosyltransferase enzymes, SSEA-1 (Lewis~ glycolipids arebiosynthesized in the brain by fucosylation of lipid substrates with the neolacto series glycolipid core structure [Galp 1-4GIcNAcp 1-3Galp 1-4GIcp I- I'Cer] (originally termed paragloboside) or its higher analogs. In order to optimize methodology for high sensitivity structural determinations of SSEA-1 type glycofipids from fetal calf brain, potential precursors and SSEA-1 glycolipids of previously established structure were first isolated from bovine erythrocytes and beige mutant mouse kidney, purified by colurnn chromatography and characterized by MALDI-TOF MS, LSIM[S, and M[S/M[S, among other techniques. Peracetylated derivatives were detected at the low ferntomole level by MALDI-TOF MS and the subnanomole level by LSIMS. MALDI-TOF MS produced mainly [M+Na]' and [M+K]' species. On the basis of the direct and tandem mass spectral analyses of peracetylated and permethylated derivatives, the carbohydrate sequences in the selected bovine erythrocyte and mouse kidney GSL fractions were found to be consistent with those of glycolipids previously reported from larger-scale studies of these sources. Their heterogeneous ceramide moieties were characterized by CID M[S/M[S of abundant Zo-type ions in the LS1 mass spectra of the permethylated GSLs. MALDI-PSD-TOF mass spectral analyses of low and subpicomole amounts of derivatized GSL fractions from fetal calf brain provided carbohydrate sequence information that indicates the presence of mono- and difucosylated SSEA-I neolacto series glycolipids and their nonfucosylated analogs.